Scientists Identify Astrocyte Dysfunction in Parkinson's Disease
By Jamie Talan
October 22, 2020
Article In Brief
Researchers have found evidence of astrocytic dysfunction in induced pluripotent stem cell-generated astrocytes from two patients with Parkinson's disease. These findings highlight a new therapeutic target and a method to model the disease in a laboratory that can also be used for drug discovery.
A team of Finnish scientists have found evidence of astrocytic dysfunction in induced pluripotent stem cell-generated astrocytes from two patients with Parkinson's disease (PD).
The findings, published online on September 2 in Nature Scientific Reports, suggest a new treatment target and a way to model the disease in a laboratory that can also be used for drug discovery.
The two patients whose cells were studied had a mutation in the leucine-rich repeat kinase 2 (LRRK2) gene; one patient also had a second mutation in the glucosylceramidase beta (GBA) gene. LRRK2 and GBA—the two most common genetic risk factors identified in PD —are found in less than 5 percent of PD patients.
Scientists have discovered that many of the genes associated with PD are highly expressed in astrocytes and play important roles in astrocytic functions. Studies of postmortem PD brains have also revealed alpha-synuclein accumulation in astrocytes. Several laboratories are now studying astrocytes' role in PD.
“The results [of the current study] provide evidence that LRRK2 and GBA mutant astrocytes are likely to contribute to PD progression and offer new perspectives for understanding the role of astrocytes in the pathogenesis of PD,” said Tuuli-Maria Sonninen, a doctoral student and a stem cell scientist at the University of Eastern Finland (UEF), who worked on the study with lead author Šárka Lehtonen, PhD, a docent in neuropharmacology at UEF and Sigrid Jusélius research fellow at the A.I. Virtanen Institute for Molecular Sciences.
“Traditionally, the focus in PD has been in dopaminergic neurons, but the role of astrocytes has not been well studied,” Sonninen told Neurology Today. “Astrocytes are the most abundant cell type in the human brain and are crucial for normal neuronal function. Recently, several studies have highlighted the role of astrocytes in several neurodegenerative diseases, including PD (showing neuroinflammation, alpha-synuclein accumulation, and reduced glutamate uptake).”
Study Methods, Findings
To explore the role of astrocytes further, the scientists collected skin fibroblasts from two people with genetic mutations for Parkinson's to make iPSC-generated astrocytes. They also obtained iPSC-generated astrocytes from two healthy volunteers. The iPSCs were generated with standard techniques: exposing the fibroblasts to Oct-4, Klf-4, Sox-2, and c-Myc, and then allowing them to differentiate into astrocytes. They then compared the response of astrocytes from the patients and healthy volunteers to a variety of tests.
Astrocytes from PD patients produced significantly higher levels of alpha-synuclein and increased levels of calcium. The scientists said that this makes sense, as others have reported that alpha-synuclein disrupts calcium homeostasis.
Next, they exposed the cells to inflammatory stimuli to measure changes in the astrocytes' response. Again, they observed significant differences between the cell lines. The PD astrocytes were more reactive to the inflammatory challenge, expressing increased cytokine levels. Further studies showed that the astrocytes also had mitochondrial disturbances and a lower mitochondrial DNA copy number. Finally, they observed increases in levels of polyamines and polyamine precursors that are involved with cell proliferation and differentiation and other cellular jobs and a decrease in lysophosphatidylethanolamine levels. They also used a CRISPR/Cas9 technique to target the LRRK2 mutation in the second PD patient and reversed the phenotype in the patient's cell lines.
The findings help explain why LRRK2 defects impair dopamine neurons, the investigators contend. They did not find significant differences between the cell lines belonging to the LRRK2 patient and the LRRK2 patient with the added GBA mutation, but there were some differences in metabolic and mitochondrial respiration between the two patients. “As with many iPSC-based studies, our study is limited with the number of patients and cell lines available for the study, and thus our results may not represent the situation in all PD patients, but rather those with LRRK2 mutations,” the scientists wrote in the paper.
“The results [of the current study] provide evidence that LRRK2 and GBA mutant astrocytes are likely to contribute to PD progression and offer new perspectives for understanding the role of astrocytes in the pathogenesis of PD.”
“Astrocytes are known to express low levels of alpha-synuclein and additionally can take up alpha-synuclein released from neurons (or from other cell types),” added Sonninen.
“Our study showed increased expression of alpha-synuclein in PD astrocytes, which is supported by other studies with iPSC-derived astrocytes. If the alpha-synuclein amount in PD astrocytes are already higher, additional alpha-synuclein from neurons can cause the aggregation of alpha-synuclein in astrocytes and dysfunction of protein degradation pathways (including autophagy). Accumulation/aggregation of alpha-synuclein in astrocytes can trigger the release of pro-inflammatory cytokines and have a toxic effect on neurons.”
The scientists are now building a microfluidic organ-on-chip model of the blood-brain barrier (BBB) using iPSC-derived endothelial cells, pericytes, and astrocytes. They will use the model to study the role of the BBB and the role of different cell types in PD. Later on, they hope to extend the model by adding neurons and microglia to study the BBB-neuro interactions in PD.
“Because of the growing evidence that other cell types (including astrocytes, microglia, and cells in BBB) in the brain are contributing to the disease progression, new in vitro models are needed which can recapitulate the brain microenvironment and include all the different cell types,” said Sonninen. “This model could be used to identify new molecular mechanisms/drug targets, and also to test and screen new drugs targeted to different cell types.”
Commenting on the study, Patrik Brundin, MD, PhD, associate director of the Van Andel Institute and director of its Center for Neurodegenerative Science in Michigan, said: “For many years, the Parkinson research field was totally focused on neurons. During the past decade, there has been a gradual realization that microglia and neuroinflammation might be important in the disease pathogenesis. Now the time has come to turn the spotlight on astrocytes.”
“A few studies in recent years have suggested that astrocytes can contribute to neurodegeneration in Parkinson's disease, and the present study adds to that body of evidence,” he continued. “It is particularly interesting that the study examines the role of LRRK2 and GBA in this context—two genes that have risk variants that elevate Parkinson risk in carriers. It may well be that these genes [contribute] to elevated Parkinson risk through actions on glia and other non-neuronal cells. This study really highlights the importance of studying non-neuronal cells (both inside and outside the brain) in the context of Parkinson risk.”
Dr. Lehtonen's group provides another important contribution that highlights the role of the protein quality control system in the pathogenesis of PD, said Amina El Ayadi, PhD, assistant professor of surgery at the University of Texas Medical Branch, who has studied astrocytic function in PD.
“For a long time, scientists have studied the death of dopamine neurons in PD with less focus on the role of surrounding glial cells like astrocytes and microglia,” Dr. Ayadi said. “These cells are at the interface between neuronal function and the systemic immune response and work to buffer the neuroinflammation response in highly stressful conditions.”
“Here again, a mutation in either LRRK2 G2019S or GBA N370S induces a decrease in the expression of the lipid metabolites that form the lysosomal membranes and interfere with the quality control and housekeeping functions ensured by autophagy. This was clearly demonstrated by increased levels of alpha-synuclein in astrocytes derived from IPSc mutants for LRRK2-G2019S and GBA-N370S, which may further contribute to protein aggregation and cell death.”
Dr. Ayadi added: “We have previously shown decreased levels of quality control proteins in the brain of Alzheimer's disease patients and observed increased neuroinflammation in stress conditions. The authors of this study make a logical correlation on how the accumulation of alpha-synuclein in astrocytes will cause the generation of pro-inflammatory agents that will lead to decreased mitochondrial function and alteration of their metabolic profile. This opens up the field for studying the role of cell metabolism in neurodegenerative diseases.”.
Sonninen and Dr. Lehtonen had no relevant disclosures. Dr. Brundin has received commercial support as a consultant from Renovo Neural, Inc., Lundbeck A/S, AbbVie, Fujifilm-Cellular Dynamics International, Axial Biotherapeutics, and Living Cell Technologies. He has also received commercial support for research from Lundbeck A/S and Roche and has ownership interests in Acousort AB and Axial Biotherapeutics.